This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Milk derived peptides have been identified as potential antihypertensive agents. The
primary objective was to investigate the effectiveness of IPP-rich milk protein hydrolysates
(MPH) on reducing blood pressure (BP) as well as to investigate safety parameters
and tolerability. The secondary objective was to confirm or falsify ACE inhibition
as the mechanism underlying BP reductions by measuring plasma renin activity and angiotensin
I and II.

Results

In subjects with stage 1 hypertension, MPH1 lowered systolic BP by 3.8 mm Hg (P =
0.0080) and diastolic BP by 2.3 mm Hg (P = 0.0065) compared with placebo. In prehypertensive
subjects, the differences in BP between MPH1 and placebo were not significant. MPH2
did not change BP significantly compared with placebo in stage I hypertensive or prehypertensive
subjects. Intake of MPHs was well tolerated and safe. No treatment differences in
hematology, clinical laboratory parameters or adverse effects were observed. No significant
differences between MPHs and placebo were found in plasma renin activity, or angiotensin
I and II.

Conclusions

MPH1, containing IPP and no minerals, exerts clinically relevant BP lowering effects
in subjects with stage 1 hypertension. It may be included in lifestyle changes aiming
to prevent or reduce high BP.

Trial registration

ClinicalTrials.gov NCT00471263

Background

High blood pressure (BP) is a controllable risk factor in the development of cardiovascular
conditions, and therefore any food component with the ability to reduce BP could contribute
to the prevention or treatment of cardiovascular diseases [1-3].

Milk-derived peptides have been identified as potential antihypertensive agents. The
best-characterized peptides found in fermented or enzymatically treated milk are Isoleucine-Proline-Proline
(IPP), and Valine-Proline-Proline (VPP). Over twenty five human studies have been
performed linking the consumption of products containing both IPP and VPP with significant
reductions in BP [4-6]. Fifteen of the BP studies have been done in Japanese subjects. Ten studies have
been performed in Caucasians, that is, in Finnish subjects [7-10], Dutch subjects [11-14], Scottish subjects [15] and American subjects [16].

Effective dosages range from 3.07 mg/d (1.60 mg IPP and 1.47 mg VPP) to 52.5 mg/d
(30 mg IPP and 22.5 mg VPP) [10,17]. Recently, casein hydrolysates with different bioactive peptide profiles were developed
based on their in vitro ACE inhibitory potential: MPH1 (or tensVida™) and MPH2. The
identified peptide contributing to ACE inhibitory effects in MPH1 is IPP, and those
in MPH2 are Methionine-Alanine-Proline (MAP), IPP, and Leucine-Proline-Proline (LPP).
In the ACE inhibition assays, MPH2 was somewhat more potent than MPH1.

An advantage is that these products contain a negligible amount of minerals contrasting
with all other lactotripeptide-based products tested so far [18-23]. Although the dosages of minerals in the sour milk are much lower than those that
were effective in lowering BP in intervention trials, and placebo treatments were
controlled for mineral content, it is possible that the minerals may have induced
additive or synergistic BP effects. To our knowledge, this is the first study performed
in Caucasian subjects using products with a negligible amount of minerals.

The primary objective of the study was to investigate the effectiveness of MPHs on
reducing BP as well as to investigate safety parameters and tolerability in subjects
with prehypertension and stage 1 hypertension. The secondary objective was to obtain
information on the mechanism of action underlying the BP effects. The dose chosen
is somewhat higher than the lower doses tested for similar products earlier in Japanese
subjects, in view of the fact that Caucasian subjects tend to show a smaller blood
pressure lowering toward lactotripeptide treatment than Japanese [5,6].

Methods

Study participants

Male and female Caucasian subjects were recruited from the pool of volunteers of TNO
Quality of Life. After being informed about the study, the subjects gave voluntary
written informed consent. Health was assessed by an interview on medical history,
physical examination, and routine laboratory tests on blood and urine. To select subjects
with prehypertension and stage 1 hypertension, the classification according to JNC-7
was used [24]: subjects with a systolic BP between 140 - 159 mm Hg or a diastolic BP between 90
- 99 mm Hg were assigned to 'stage 1 hypertension', and subjects with a systolic BP
between 120 - 139 mm Hg or a diastolic BP between 80 - 89 mm Hg were designated 'prehypertension'.
BP was measured on three separate visits. Subjects with a history of medical events
or medication that may have influenced the study outcome were excluded from participation.
Subjects who were on slimming, medically prescribed, vegan, vegetarian or macrobiotic
diet were also excluded as were smokers and subjects using more than 28 units (men)
or 21 units (women) of alcohol per week to exclude alcoholics. Pregnant or lactating
women were also excluded from participation. Eighty healthy subjects were included
in the study.

Study design

The study was designed as a randomized, placebo-controlled, double blind, crossover
study. Study treatments were given to the subjects according to a Latin square design.
In view of the very short half-life of lactotripeptides [25], no wash-out period was included between treatments. The study was powered to detect
a 3.5 mmHg systolic blood pressure difference between placebo and treatment at an
α of 0.05 (two-sided) and a β of 0.8.

Study protocol

Subjects visited the test facility of TNO Quality of Life at three consecutive test
days at the end of each treatment period. At the first two test days, BP measurements
were performed two hours after the subjects had their own habitual breakfast and the
study substances at home. At the third test day, subjects came in a fasted state for
collection of blood and spot-urine samples. No alcohol and sports were allowed the
day before the test days, and no dairy products were allowed at breakfast on the test
days. During the study period, subjects were instructed not to consume more than one
portion of a fermented dairy product per day.

The study was performed according to the ICH Guideline for Good Clinical Practice
(ICH topic E6, adopted 01-05-1996 and implemented 17-01-1997) and was approved by
the independent Medical Ethics Committee METOPP (Tilburg, the Netherlands).

Study products

MPH1 and MPH2 were produced through hydrolysis of glycomacropeptide and casein, respectively.
Hydrolysis was performed using a proline-specific endoprotease. The nutritional properties,
as well as the amounts of bioactive peptides of the study products are presented in
table 1. Lactotripeptides were quantified as described in [26].

Study treatments

Study treatments were offered to the volunteers in capsules, to exclude any pre-ingestion
interference with food matrices. The treatments consisted of consumption of MPH1 or
MPH2 or placebo during 4 weeks. Five hundred mg hydrolysed protein was used per capsule
for each of the MPH treatments. The lactotripeptide content per capsule was 7.5 mg
IPP for MPH1, and 6.6 mg MAP, 2.3 mg LPP, and 1.8 mg IPP for MPH2. Subjects received
2 capsules per day, and were instructed to consume one capsule directly upon completion
of breakfast and one capsule directly upon completion of dinner. Placebo treatment
consisted of daily consumption of 2 capsules, each of them filled with 500 mg cellulose
(food grade quality).

Blood pressure measurements

BP was measured between 2.5 and 3.5 h after ingestion of the study substances using
automated digital sphygmomanometry (OMRON IC). In brief, subjects rested for at least
15 min at room temperature. The BP cuff was placed around the upper left arm which
was supported at the level of the heart while the subject sat in a chair with the
feet positioned flat on the floor and with a straight back rested against the chair.
The first measurement was taken after three minutes rest. The subject was told not
to move or speak. In total four measurements were taken with one minute rest in between.
The average of the last three measurements was calculated.

Safety parameters

Clinical laboratory tests were performed at screening and at the end of treatment
and included hematology, serum chemistry (liver enzymes, albumin, bilirubin, urea,
creatinin, cholesterol parameters, triacylglycerols, glucose, minerals, blood sedimentation
rate, and C-reactive protein). Blood samples were obtained from the antecubital vein
of the forearm and collected in tubes containing clot activator for serum and in tubes
containing potassium ethylene diamine tetra acid (K3EDTA) for plasma (Vacutainer Systems, Becton Dickinson, Plymouth, UK). Blood was centrifuged
for 15 min at ca. 2,000 × g at ca. 4°C within 15-30 min after collection, and stored
at -20°C until analysis. All biochemical determinations in blood were performed at
TNO Quality of Life using Olympus AU400 analytical equipment and reagents. Dipstick
urinalysis was performed on the day of collection to assess the presence of protein,
glucose, leucocytes, erythrocytes, nitrite, pH, ketones, bilirubin, and urobilinogen.
A microscopic inspection of sediment was done if the dipstick test gave values above
the normal range for leucocytes, blood or protein.

At the end of treatment, subjects indicated the presence or absence of a number of
possible adverse effects in a questionnaire using a 4-point scale from 'not at all'
to 'very often'. Symptoms included not feeling well, headache, weakness, fatigue,
nausea, vomiting, burping, stomach-ache, bloating, flatulence, constipation, diarrhea,
dry mouth, change of taste, cough, and skin complaints.

Renin was measured by a standard immunoradiometric assay (Schering SA, CIS bio international,
France). Intra- and inter-assay coefficients of variation were 4.1% and 7.6%, respectively.
Angiotensin I and II were measured by a standard radioimmunoassay (Peninsula Laboratories,
St. Helens, UK) following C-18 Sep-Pak (Waters-Millipore) extraction of the peptide.
Intra- and inter-assay coefficients of variation were 4.6% and 7.7%, respectively.

Statistical analysis

For blood pressure, for every subject and treatment, measurements were first averaged
per test day, and subsequently averaged over two successive test days. Statistical
analysis of treatment effects on mean DBP and SBP and on mechanistic parameters was
performed taking into account the study population, BP class (i.e. prehypertension
or stage 1 hypertension), treatment, and interaction between treatment and BP class.
Treatment effects were investigated using 2-way ANOVA. Carry over analysis was performed
to determine whether a carry over correction was necessary. In case no significant
carry over effect was found, the model was simplified by removing this effect. If
the analysis of variance indicated an overall treatment effect (P < 0.05), comparisons
between treatment means were performed using a 2-sided (paired) Student's t-test.
Dichotomized scores in the questionnaire on possible adverse-effects were analyzed
using Cochran-Mantel-Haenszel statistics taking into account multiple measurements
per subject. In all statistical tests, the null hypothesis was rejected at the 0.05
level of probability (α = 5%). All data are presented as mean ± standard deviation
(SD). Statistical analysis of the data was carried out using the SAS statistical software
package (SAS/STAT Version 8.2, SAS Institute, Cary, NC).

Results

Study participants

Eighty healthy subjects (48 men, 32 women) were included with a mean age of 58 ± 8
y, body mass index (BMI) of 26.2 ± 3.1 kg/m2, systolic/diastolic BP of 136.2 (±12.7)/84.0
(±8.8) mm Hg. Thirty-four subjects had stage 1 hypertension with a mean systolic/diastolic
BP of 147.0 (±11.7)/91.5 (±6.1) mm Hg, and 46 subjects had prehypertension with a
mean systolic/diastolic BP of 128.1 (±6.1)/78.5 (±6.1) mm Hg. Upon data analysis,
placebo BP values were found to be lower than baseline values at the time of screening.
Because of this, a number of subjects fell into a lower BP class compared to the onset
of the study. As there was no effect of treatment order on BP decrease of either intervention,
subjects were classified as having prehypertension or stage 1 hypertension based on
placebo values for further analysis of the treatment effects. There was no significant
difference between treatments concerning the number of reclassified subjects. This
procedure resulted in 26 subjects having stage 1 hypertension, 44 subjects having
prehypertension, and 10 subjects having a normal BP (systolic BP < 120 mm Hg and diastolic
BP < 80 mm Hg). These normotensives were excluded from further analyses, because the
number of subjects was too small for a sound evaluation. Baseline characteristics
of the study population (n = 70) are shown in Table 2. Compliance with intake of the study substances, assessed by counting returned capsules,
was very good. Also compliance with study procedures with respect to maintenance of
habitual diet and physical activity pattern was very good.

Blood pressure

The BP values after 4 weeks treatment are presented in Table 3. The data indicate that in stage 1 hypertensive subjects, MPH1 induced a significant
lowering of systolic BP by 3.8 mm Hg (P = 0.0080) and of diastolic BP by 2.3 mm Hg
(P = 0.0065) compared with placebo. In subjects with prehypertension, the differences
in systolic BP and diastolic BP between MPH1 and placebo were not significant. MPH2
did not change BP significantly as compared with placebo in either stage I hypertensives
or prehypertensives.

Table 3. Blood pressure measurements in subjects with stage 1 hypertension and in subjects
with prehypertension after daily intake of a placebo, or MPH 1 or MPH 2 for 4 weeks1

Safety parameters

Intake of both MPHs was well tolerated. No significant differences in clinical laboratory
parameters and adverse events were observed between placebo and MPHs.

Thirty seven subjects reported a adverse effect after intake of the placebo (in total
118 occurrences of adverse effects), 43 subjects reported a adverse effect after intake
of MPH1 (in total 126 occurrences of adverse effects), and 37 subjects reported a
adverse effect after consumption of MPH2 (in total 114 occurrences of adverse effects).
Most reported symptoms occurred 'hardly ever' or 'sometimes'. In single occasions,
a symptom occurred 'often' or 'very often', but no clear differences were observed
between treatments. Adverse effects most often reported were flatulence, headache,
dry mouth, and fatigue. Only flatulence was significantly more often reported after
placebo treatment (23×) compared with MPH1 (16×; P = 0.0196) and MPH2 (14×; P = 0.0339)
(Figure 1). Since the frequency and seriousness of the reported adverse effects was low, the
above mentioned differences between MPHs and placebo were not considered clinically
relevant.

Mechanistic parameters

No significant differences between MPHs and placebo treatment were found in plasma
renin activity or concentrations of angiotensin I and II (Figure 2). In all subjects, intake of the placebo, MPH1, and MPH2 for 4 weeks resulted in
similar mean renin activities. Mean angiotensin I and II concentrations after intake
of placebo were comparable with those after intake of MPH1 and MPH2.

Discussion

In subjects with stage 1 hypertension, daily intake of MPH1, delivering 15 mg IPP,
for 4 weeks lowered systolic BP by 3.8 mm Hg and diastolic BP by 2.3 mm Hg compared
with placebo. Daily intake of MPH2 containing 13.2 mg MAP, 4.6 mg LPP, and 3.7 mg
IPP did not affect BP in these subjects. Since MPH1 contains primarily IPP while MPH2
contains mainly MAP and LPP, this differential effect despite equal protein dose of
the two treatments suggests superiority of IPP over the other peptides for BP lowering,
or even functional antagonism of MAP and LPP against IPP (i.e. MAP and LPP having
an opposite effect compared to IPP in vivo). Alternatively, MAP and LPP may be less
bioavailable than IPP. The bioavailability of IPP has been demonstrated in humans
[25], but bioavailability data for MAP and LPP are lacking.

In our study MPHs were given for a period of 4 weeks. A further lowering of BP might
have been achieved in a longer treatment periods as has been shown in studies on comparable
products [27-30].

In subjects with prehypertension, daily intake of both MPH1 and MPH2 did not affect
BP. Also in other studies, lactotripeptides, in particular VPP and IPP, appear more
effective in reducing BP of subjects with a higher starting BP (Sano et al, 2005;
Aihara et al, 2005). Indeed, in none of the trials with normotensives were any statistically
significant BP changes found [31-34]. This is in line with findings in studies using pharmaceutical interventions [35].

In many papers, BP reduction through lactotripeptides has been suggested to be due
to inhibition of the renin-angiotensin system, which plays an important role in regulating
arterial pressure. Renin converts angiotensinogen to the biologically inactive angiotensin
I, which in turn undergoes proteolytic cleavage by ACE to the vasoconstrictor angiotensin
II [36]. Inhibition of ACE leads to an increase of the angiotensin I/angiotensin II ratio
and a subsequent compensatory increase in renin activity. We could not demonstrate
any effects of either MPH on renin activity, angiotensin I and II. Although local
ACE inhibitory effects near the vascular wall may be overlooked by measuring angiotensin
in blood samples from the systemic circulation, these findings do not support an angiotensin-mediated
effect of ACE inhibition in vivo. This may explain why the in vitro ACE inhibitory
potency of MPH1 and MPH2 did not predict their BP lowering activity. Indeed, based
on their IC50 values, MPH2 was expected to lower BP more than MPH1, but the contrary
was found in this study.

An alternative way in which ACE inhibition could be involved in the observed BP effects
would be through an increased availability of the endogenous vasodilator bradykinin
[37]. Alternative mechanisms, such as such as opioid-like activities, inhibition of the
release of the vasoactive substances such as the vasoconstrictor endothelin-1, eicosanoids
and nitric oxide have been proposed to underlie the blood pressure lowering effect
of lactotripeptides (see [38] for a review). Further work is needed to better characterize these mechanisms.

Since lactotripeptides are frequently taken in a dairy drink or a similar vehicle,
it is often unclear whether the blood pressure lowering effects are solely due to
the lactotripeptides, or whether minerals in these drinks contribute to the blood
pressure lowering effects as well. In the current study, the lactotripeptides have
been administered in capsules rather than in dairy drinks. Also, MPH1 contains very
few minerals (1.1 mg K+ + Ca2+ per mg IPP) compared to other products that have been administered in capsules in
other studies (2.3 - 3.6 mg K+ + Ca2+ per mg IPP) [28,31,33,39]. Therefore, it is very unlikely that minerals have contributed to the observed blood
pressure lowering effect.

The doses of MPH used in this study did not exert any significant effects on blood
and urine parameters, adverse events, and other adverse effects, and were thus considered
safe. Previous studies confirmed that even high daily dosages of VPP and IPP were
safe [10,31,33,34,39].

The BP reductions found for MPH1 are in the order of magnitude of those that can be
achieved by lifestyle modifications, such as weight loss in overweight subjects [40], regular aerobic exercise [41,42], adjustment of dietary habits [43], and moderation of alcohol consumption [44]. Cook and co-workers [45] and Whelton and co-workers [41] investigated the impact of small reductions in the population distribution of diastolic
BP, such as those found in our study and those potentially achieved by lifestyle modification,
on incidence of cardiovascular heart disease and stroke. Cook estimated that a 2 mm
Hg reduction in the population average of diastolic BP for white US subjects aged
35-64 y would result in a 17% decrease in the prevalence of hypertension, a 15% reduction
in the risk of stroke, and a 6% reduction in the risk of coronary heart disease.

Conclusions

MPH1, an IPP-rich milk protein hydrolysate, is safe and exerts relevant BP lowering
effects in subjects with stage 1 hypertension. It may be included in lifestyle changes
aiming to prevent or reduce high BP.

Competing interests

E.B. did not have a conflict of interest. J.K. is employed as a senior scientist at
the Department of Nutrition and Health at DSM Biotechnology Center, the Netherlands.

The study was financially supported by DSM Food Specialties, Delft, the Netherlands,
the manufacturer of the study substances.

Authors' contributions

EB and JK were responsible for design of the study; EB was responsible for conduct
of the study and data interpretation; EB and JK wrote the manuscript; Both JK and
EB read and approved the final version.

Acknowledgements

We thank the persons of TNO Quality of Life assisting in the organization, conduct
and completion of the study. We thank Carina Rubingh and Sabina Bijlsma for statistical
analyses, and Paul Schiffers for analyses of mechanistic parameters. We thank the
volunteers for their participation.